Hhla2 as a novel inhibitor of human immune system and uses thereof

ABSTRACT

Provided are methods of treating an autoimmune disease in a subject, or of suppressing transplant rejection in a subject, or of treating a cancer in a subject, as well as compositions therefor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/121,771, filed Sep. 5, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/770,120, filed Aug. 25, 2015, now U.S. Pat. No.10,093,737, issued on Oct. 9, 2018, which is a U.S. National Stage ofPCT International Patent Application No. PCT/US2014/015308, filed Feb.7, 2014, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/771,305, filed Mar. 1, 2013, the contents of each of which areherein incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbers1DP2DK083076 awarded by the National Institutes of Health and PC094137awarded by the Department of Defense. The government has certain rightsin the invention.

SEQUENCE LISTING

This application contains a Sequence Listing, which was submitted inASCII format via Patent Center, and is hereby incorporated by referencein its entirety. The ASCII copy, created on Jan. 24, 2022, is nameSequence Listing.txt and is 12 KB in size.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparentheses. Full citations for these references may be found at the endof the specification. The disclosures of these publications, and allpatents, patent application publications and books referred to herein,are hereby incorporated by reference in their entirety into the subjectapplication to more fully describe the art to which the subjectinvention pertains.

Interactions between members of the B7 ligand and CD28 receptor familiesgenerate positive costimulation and negative coinhibition, which are ofcentral importance in regulating T cell responses (1-3).B7-1/B7-2/CD28/CTLA-4 is the most extensively characterized of thesepathways. Ligands B7-1 (CD80) and B7-2 (CD86) on antigen-presentingcells (APCs) bind to CD28 on naïve T cells, and provide a majorcostimulatory signal to activate naive T cells. After the initialactivation, coinhibitory molecule cytotoxic T lymphocyte antigen-4(CTLA-4, CD152) is induced on T cells and engages the same B7-1 and B7-2ligands in order to restrain T cell function. In contrast to thecostimulatory activity of CD28, the interaction of B7-1 or B7-2 withCTLA-4 is essential for limiting the proliferative response of recentlyactivated T cells to antigen and CD28-mediated costimulation.

During the past decade, several new pathways in the B7 and CD28 familieshave been identified, including B7h/ICOS, PD-L1/PD-L2/PD-1,B7-H3/receptor and B7x/receptor. B7h (4) (also called ICOS-L, B7RP-1(5),GL50(6), B7H2(7), LCOS(8), and CD275) binds to the induciblecostimulator (ICOS, CD278) on activated T cells (9), which inducesstrong phosphatidylinositol 3-kinase activity (10, 11) and leads to theexpression of transcription factors involved in follicular helper CD4 T(Tfh) differentiation (12). Therefore, the B7h/ICOS pathway providescritical T cell help to B cells. Deficiencies in this pathway result insubstantially reduced numbers of memory B cells and markedly reducedlevels of serum Ig in patients with common variable immunodeficiency(13). In humans, but not in mice, B7h can bind both CD28 and CTLA-4(14). The B7 family members PD-L1 (15) (also termed B7-H1(16), CD274)and PD-L2 (17) (also called B7-DC(18), CD273) bind to the programmeddeath 1 receptor (PD-1, CD279), which ultimately decreases induction ofcytokines and cell survival proteins in T cells. The PD-L/PD-1 pathwayplays an important role in the control of tolerance and autoimmunity(19), and contributes critically to T cell exhaustion and viralpersistence during chronic infections (20). In addition, PD-L1 can alsobind to B7-1(21).

B7-H3 (22)(CD276) and B7x (23) (also called B7-H4 (24) or B7S1 (25)) arerecently discovered members of the B7 family, and their contributions toimmune response have not yet been clearly defined. B7-H3 binds activatedT cells, but the physiological role of this pathway is unclear, as bothcostimulatory and coinhibitory effects have been observed (26). B7xbinds activated T cells and inhibits T cell functions. In addition,myeloid derived suppressor cells (MDSCs) also express a receptor for B7x(27). Clinical data also support a coinhibitory function for B7x, asaberrant expression of this molecule is observed in many types of humancancers and is often associated with enhanced disease progression andpoor clinical outcome (28). It appears that the B7x pathway is exploitedas part of the immune evasion mechanisms used by many human cancers.Collectively, the regulated spatial and temporal expression ofcostimulatory and coinhibitory B7 molecules provides the controls thatunderlie T cell-mediated immune responses.

Due to their fundamental biological importance and therapeuticpotential, there has been considerable interest in the identification ofadditional molecules with costimulatory or coinhibitory function.

The present invention addresses the need for improved therapies andtherapeutics based on a novel B7/CD28 family pathway, namely, a HERV-HLTR Associating Protein 2 (HHLA2) pathway.

SUMMARY OF THE INVENTION

A method is provided of treating an autoimmune disease in a subject, orof suppressing transplant rejection in a subject, comprisingadministering to the subject an amount of an HHLA2-immunoglobulin-fusionprotein effective to treat an autoimmune disease, or suppress transplantrejection, respectively.

Also provided is a method of treating a cancer in a subject, comprisingadministering to the subject an amount of an anti-HHLA2 antibody, or anHHLA2-binding fragment of an anti-HHLA2 antibody, effective to treat acancer.

Also provided is a method of treating an infectious disease a subject,comprising administering to the subject an amount of an anti-HHLA2antibody, or an HHLA2-binding fragment of an anti-HHLA2 antibody,effective to an infectious disease.

Also provided is a composition comprising an HHLA2-immunoglobulin-fusionprotein and a pharmaceutically acceptable carrier.

Also provided is a composition comprising an anti-HHLA2 antibody, or anHHLA2-binding fragment of such an antibody, and a pharmaceuticallyacceptable carrier.

Also provided is an HHLA2-immunoglobulin-fusion protein for treating anautoimmune disease in a subject, or for suppressing transplant rejectionin a subject.

Also provided is an anti-HHLA2 antibody, or an HHLA2-binding fragment ofsuch an antibody, for treating a cancer in a subject.

Also provided is a screening method for identifying an agent as atreatment for an autoimmune disease in a subject, or as a treatment forsuppressing transplant rejection in a subject, comprising contacting animmune system cell expressing an HHLA2 receptor with the agent andquantifying activity of the cell in the presence and the absence of theagent, and identifying the agent as a treatment for an autoimmunedisease in a subject, or as a treatment for suppressing transplantrejection in a subject, wherein an agent that inhibits the activity ofthe cell in its presence as compared to in its absence is indicated tobe a treatment for an autoimmune disease in a subject or a treatment forsuppressing transplant rejection in a subject, and an agent that doesnot affect activity of, or stimulates activity of , the cell itspresence as compared to in its absence is not indicated to be atreatment for an autoimmune disease in a subject or a treatment forsuppressing transplant rejection in a subject.

Also provided is a screening method for identifying an agent as atreatment for a cancer or for an infectious disease, comprisingcontacting an HHLA2 receptor with the agent and an amount of HHLA2 andquantifying the antagonism of the agent on HHLA2 binding to the HHLA2receptor, and identifying the agent as a treatment for a cancer or foran infectious disease,

wherein an agent that competes with, and/or reduces, HHLA2 binding tothe HHLA2 receptor in its presence as compared to in its absence isindicated to be a treatment for a cancer or an infectious disease, andan agent that does not affect HHLA2 binding in its presence as comparedto in its absence is not indicated to be a treatment for a cancer or foran infectious disease. In an embodiment, the HHLA2 receptor is expressedon a cell. In an embodiment, the cell is an immune system cell. In anembodiment, the cell is CD4 T cell, a CD9 T cell or anantigen-presenting cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C. HHLA2 is a member of the B7 family and forms a group withB7x and B7-H3. (A) Predicted signal peptide, IgV-like and IgC-likedomains, transmembrane region, and cytoplasmic tail of human HHLA2protein were indicated. The potential N-glycosylation sites werearrowed. (B) Confocal microscopy showed that human HHLA2-YFP protein(YFP=Yellow Fluorescent protein) was predominantly expressed on cellmembranes of the 3T3 cells, whereas human CTLA-4-YFP fusion protein wasmainly localized intracellularly in the 3T3 cells. (C) Phylogenetic treeof the human B7 family. The phylogenetic comparison of human B7molecules was generated by PAUP version 4.0b10. The family was dividedinto three groups: HHLA2, B7x, and B7-H3 for group III; PD-L1 and PD-L2for group II; B7-1, B7-2, and B7h for group I. Receptors for human B7molecules are also indicated.

FIG. 2A-2B. Analysis of endogenous HHLA2 protein expression by flowcytometry with specific mAb. (A) 3T3 or CT26 cells were transfected withMSCV vectors to stably express cell surface human B7-1, B7-2, B7h,PD-L1, PD-L2, B7-H3, B7x, and HHLA2-YFP. Transfectants were stained withan anti-HHLA2 mAb clone 566.1 (open histograms) or isotype control(shaded histograms) for FACS. (B) Human peripheral blood mononuclearcells (PBMCs) were stained with biotin-anti-HHLA2 mAb/APC-streptavidin,and PE-, FITC-, or Percp-Cy5.5-conjugated anti-CD14 (monocytes),anti-CD19 (B cells), anti-CD4, anti-CD8, and anti-PD-L1. Monocytes and Bcells were activated with LPS/IFN-γ for three days, whereas T cells wereactivated with anti-CD3 for three days. Immature dendritic cells (DCs)were generated from blood monocytes incubated with GM-CSF/IL-4 and wereinduced with LPS/IFN-γ to become mature DCs. Endogenous HHLA2 proteinwas highly detected on monocytes and induced on B cells, whereas PD-L1was induced only on activated immune cells. Anti-HHLA2 mAb (openhistograms) and isotype control (shaded histograms). Representative ofat least seven experiments.

FIG. 3A-3C. HHLA2 does not bind other known members of the CD28 and B7families. (A) 3T3 or CT26 cells were transfected with MSCV vectors tostably express cell-surface human CD28, CTLA-4 without cytoplasmic tail,ICOS, PD-1, B7-1, B7-2, B7h, PD-L1, PD-L2, B7-H3, and B7x. Alltransfectants were stained with specific mAbs (open histograms) orcontrol Abs (shaded histograms). (B) Transfectants were stained withHHLA2-Ig fusion protein (open histograms) or control fusion proteins Igor B7x-Ig (shaded histograms), and then stained with a PE-conjugateanti-human IgG Fc. (C) As positive controls, 3T3 cells expressing PD-L1or PD-L2 were stained with PD-1-Ig (open histograms) or control Ig(shaded histograms).

FIG. 4A-4B. T cells and other immune cells constitutively express aputative receptor for HHLA2. (A) T cells, B cells and monocytes fromPBMCs and DCs derived from blood monocytes were stained with HHLA2-Igfusion protein (open histograms) or control Ig (shaded histograms), andthen stained with a PE-conjugate anti-human IgG Fc. CD4 and CD8 T cellswere stimulated with anti-CD3 for three days, whereas B cells andmonocytes were stimulated with LPS/IFN-γ for three days. Immature DCswere generated from blood monocytes and were induced with LPS/IFN-γ tobe mature DCs. HHLA2 bound T cells, B cells, monocytes and DCs. ICOS wasinduced on activated CD4 and CD8 T cells, whereas PD-L1 was induced onAPCs. (B) In contrast to these immune cells, HHLA2 bound neither humanHeLa cells nor mouse 3T3 cells. HHLA2-Ig fusion protein (openhistograms) and control Ig (shaded histograms). Representative of atleast five experiments.

FIG. 5A-5C. Coinhibition of HHLA2 on TCR-mediated CD4 and CD8 T cellproliferation. (A) T cells purified from PBMCs were activated with acombination of plate-bound anti-CD3 and either plate-bound HHLA2-Ig (4m/ml), control Ig (4 m/ml), or B7x-Ig (4 μg/ml) for three days.Metabolic activity was then determined by MTT assay. (B-C) CFSE-labeledT cells were stimulated with a combination of plate-bound anti-CD3 andeither plate-bound HHLA2-Ig (10 μg/ml), control Ig (10 m/ml), or B7x-Ig(10μg/m1) for five days. T cells were then stained with anti-CD4 andanti-CD8, and analyzed by flow cytometry. Representative FACS plotsshowed CFSE dilution among CD4 and CD8 T cells (B). The percentages ofproliferating CD4 and CD8 T cells were calculated by CFSE dilution (C).N=9-12, *P<0.05; **P<0.01, ***P<0.001

FIG. 6 . Inhibition of HHLA2 on cytokine production from T cells.Purified T cells were stimulated with a combination of plate-boundanti-CD3 and either plate-bound HHLA2-Ig (4 m/ml) or control Ig (4 m/ml)for three days. The cytokine levels of the supernatants were measuredusing Th1/Th2/Th9/Th17/Th22 flowcytomix. HHLA2 significantly reducedproduction of seven cytokines from T cells including IFN-γ, TNF-α, IL-5,IL-10, IL-13, IL-17A, and IL-22. N=24, **P<0.01, ***P<0.001

FIG. 7 : Immunohistochemistry results showing HHLA2 is over-expressed in50% or more of the cancerous lung, thyroid, breast, ovary, pancreas, andmelanoma samples and is also expressed in other cancerous esophagus,kidney, liver, bladder, and prostate.

DETAILED DESCRIPTION OF THE INVENTION

A method is provided of treating an autoimmune disease in a subject, orof suppressing transplant rejection in a subject, comprisingadministering to the subject an amount of an HHLA2-immunoglobulin-fusionprotein effective to treat an autoimmune disease, or suppress transplantrejection, respectively.

In an embodiment, the method is of treating an autoimmune disease.

As used herein, autoimmune diseases treatable by the method includeacute disseminated encephalomyelitis (ADEM), alopecia areata,antiphospholipid syndrome, autoimmune cardiomyopathy, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease,autoimmune lymphoproliferative syndrome, autoimmune peripheralneuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome,autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura,autoimmune urticarial, autoimmune uveitis, Behcet's disease, celiacdisease, Chagas disease, cold agglutinin disease, Crohn's disease,dermatomyositis, diabetes mellitus type 1, eosinophilic fasciitis,gastrointestinal pemphigoid, Goodpasture's syndrome, Grave's syndrome,Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto'sthyroiditis, lupus erythematosus, Miller-Fisher syndrome, mixedconnective tissue disease, myasthenia gravis, pemphigus vulgaris,pernicious anaemia, polymyositis, psoriasis, psoriatic arthritis,relapsing polychondritis, rheumatoid arthritis, rheumatic fever,Sjogren's syndrome, temporal arteritis, transverse myelitis, ulcerativecolitis, undifferentiated connective tissue disease, vasculitis, andWegener's granulomatosis.

In an embodiment, the autoimmune disease treated is adult rheumatoidarthritis.

In an embodiment, the method is of suppressing transplant rejection. Themethod treats graft versus host disease, for example where stem cells orbone marrow is transplanted. In an embodiment, the transplant rejectionsuppressed is a kidney transplant rejection. In an embodiment, thetransplant rejection suppressed is a lung, heart, pancreas, cornea, orliver transplant rejection.

In an embodiment, the HHLA2-immunoglobulin-fusion protein comprises anextracellular domain of human HHLA2. In an embodiment, the extracellulardomain comprises the sequence set forth in SEQ ID NO:1 but excluding thesignal peptide, transmembrane domain and cytoplasmic tail. In anembodiment, the HHLA2-immunoglobulin-fusion protein comprises a humanIgG Fc region. In an embodiment, the HHLA2-immunoglobulin-fusion proteincomprises a human IgM Fc region. In an embodiment, the IgG is an IgG1.In an embodiment, the HHLA2-immunoglobulin-fusion protein comprises theconsecutive amino acid residues set forth in SEQ ID NO:2.

In an embodiment, the methods can be performed, mutatis mutandis,wherein a nucleic acid encoding an HHLA2-Ig is administered in a fashionsuch that it can express inside the subject, in place of the HHLA2-Igfusion protein. In an embodiment of this method, the nucleic acidsequence comprises the following (SEQ ID NO:3):

atgaagttatgcatattactggccgtcgtggcctttgttggcctctcgctcgggagatctttggctttcttcatttatgttcctatgaatgaacaaatcgtcattggaagacttgatgaagatataattctcccttcttcatttgagaggggatccgaagtcgtaatacactggaagtatcaagatagctataaggttcatagttactacaaaggcagtgaccatttggaaagccaagatcccagatatgcaaacaggacatcccttttctataatgagattcaaaatgggaatgcgtcactatttttcagaagagtaagccttctggacgaaggaatttacacctgctatgtaggaacagcaattcaagtgattacaaacaaagtggtgctaaaggtgggagtttttctcacacccgtgatgaagtatgaaaagaggaacacaaacagcttcttaatatgcagcgtgttaagtgtttatcctcgtccaattatcacgtggaaaatggacaacacacctatctctgaaaacaacatggaagaaacagggtctttggattctttttctattaacagcccactgaatattacaggatcaaattcatcttatgaatgtacaattgaaaattcactgctgaagcaaacatggacagggcgctggacgatgaaagatggccttcataaaatgcaaagtgaacacgtttcactctcatgtcaacctgtaaatgattatttttcaccaaaccaagacttcaaagttacttggtccagaatgaaaagtgggactttctctgtcctggcttactatctgagctcctcacaaaatacaattatcaatgaatcccgattctcatggaacaaagagctgataaaccagagtgacttctctatgaatttgatggatcttaatctttcagacagtggggaatatttatgcaatatttcttcggatgaatatactttacttaccatccacacagtgcatgtagaaccgagccaagaaacagcttccggcggccgctctaaaactagtggatccgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcct ctccctgtctccgggtaaa.

In an embodiment, of all aspects of the invention described hereinreciting a subject, the subject is a human. In an embodiment, of allaspects of the invention described herein reciting HHLA2, the HHLA2 is ahuman HHLA2. In an embodiment, of all aspects of the invention describedherein reciting an anti-HHLA2 antibody (or fragment thereof), the HHLA2to which the antibody is directed (targeted) is a human HHLA2.

In an embodiment, the HHLA2 is a human HHLA2 comprising the followingsequence (SEQ ID NO:1)

MKAQTALSFFLILITSLSGSQGIFPLAFFIYVPMNEQIVIGRLDEDIILPSSFERGSEVVIHWKYQDSYKVHSYYKGSDHLESQDPRYANRTSLFYNEIQNGNASLFFRRVSLLDEGIYTCYVGTAIQVITNKVVLKVGVFLTPVMKYEKRNTNSFLICSVLSVYPRPIITWKMDNTPISENNMEETGSLDSFSINSPLNITGSNSSYECTIENSLLKQTWTGRWTMKDGLHKMQSEHVSLSCQPVNDYFSPNQDFKVTWSRMKSGTFSVLAYYLSSSQNTIINESRFSWNKELINQSDFSMNLMDLNLSDSGEYLCNISSDEYTLLTIHTVHVEPSQETASHNKGLWILVPSAILAAFLLIWSVKCCRAQLEARRSRHPADGAQQERCCVP PGERCPSAPDNGEENVPLSGKV

In an embodiment, the HHLA2-immunoglobulin-fusion protein is anHHLA2-IgG comprising the following sequence (SEQ ID NO:2)

RSLAFFIYVPMNEQIVIGRLDEDIILPSSFERGSEVVIHWKYQDSYKVHSYYKGSDHLESQDPRYANRTSLFYNEIQNGNASLFFRRVSLLDEGIYTCYVGTAIQVITNKVVLKVGVFLTPVMKYEKRNTNSFLICSVLSVYPRPIITWKMDNTPISENNMEETGSLDSFSINSPLNITGSNSSYECTIENSLLKQTWTGRWTMKDGLHKMQSEHVSLSCQPVNDYFSPNQDFKVTWSRMKSGTFSVLAYYLSSSQNTIINESRFSWNKELINQSDFSMNLMDLNLSDSGEYLCNISSDEYTLLTIHTVHVEPSQETASGGRSKTSGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK.

In an embodiment, an HHLA2 receptor as described herein has the same, orhas 99% or more sequence identity to, an HHLA2 receptor on a human Tcell or a human antigen presenting cell.

Also provided is a method of treating a cancer in a subject, comprisingadministering to the subject an amount of an anti-HHLA2 antibody, or anHHLA2-binding fragment of an anti-HHLA2 antibody, effective to treat acancer.

There are at least two mechanisms by which an anti-HHLA2 antibody, orHHLA2-binding fragment thereof, and compositions comprising such, cantreat a cancer: 1) the antibody or fragment can block the inhibition ofHHLA2 expressed on immune cells; and 2) the antibody or fragment canblock the inhibition of HHLA2 expressed on cancer cells. These twomechanisms can exist at the same time. Moreover, the cancer cells, suchas tumor cells, do not need to be HHLA2 positive.

Cancers, including tumors, treatable by the invention include of thenasopharynx, pharynx, lung, bone, brain, sialaden, stomach, esophagus,testes, ovary, uterus, endometrium, liver, small intestine, appendix,colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast,cervix, vagina, vulva, prostate, thyroid, skin, or is a glioma. In anembodiment, the cancer treated is a metastatic melanoma. In anembodiment, the cancer treated comprises a tumor. In an embodiment, thecancer treated comprises a HHLA2-bearing tumor.

A method is provided for treating a cancer in a subject comprisingadministering to the subject an anti-HHLA2 antibody, or antigen-bindingfragment thereof, or HHLA2-binding aptamer, or isolated HHLA2 receptorprotein, in an amount effective to treat a cancer in a subject. In anembodiment, the subject is a human subject. In an embodiment, theanti-HHLA2 antibody, or antigen-binding fragment thereof, orHHLA2-binding aptamer, or isolated HHLA2 receptor protein, has acytotoxic agent attached thereto. In an embodiment, the anti-HHLA2antibody, or antigen-binding fragment thereof, or HHLA2-binding aptamer,has an anti-cancer drug conjugated thereto. In an embodiment, theanti-HHLA2 antibody, or antigen-binding fragment thereof, orHHLA2-binding aptamer, or isolated HHLA2 receptor protein, has achemotherapeutic agent attached thereto. In an embodiment, theantigen-binding fragment of an anti-HHLA2 antibody is administered. Inan embodiment, the antigen-binding fragment of an anti-HHLA2 antibody isadministered as a fusion protein. In an embodiment, the isolated HHLA2receptor protein is administered. In an embodiment, the isolated HHLA2receptor protein is administered as a fusion protein. In an embodiment,the fusion protein comprises the antigen-binding fragment of ananti-HHLA2 antibody bound via a peptide bond to an immunoglobulin Fcregion. In an embodiment, the immunoglobulin Fc region is animmunoglobulin G Fc region or an immunoglobulin M Fc region. In anembodiment, the fusion protein comprises the isolated HHLA2 receptorprotein bound via a peptide bond to an immunoglobulin Fc region. In anembodiment, the immunoglobulin Fc region is an immunoglobulin G Fcregion or an immunoglobulin M Fc region. In an embodiment, theanti-HHLA2 antibody, or antigen-binding fragment thereof, isadministered as a mammalian dendritic cell loaded with the anti-HHLA2antibody, or antigen-binding fragment thereof. In an embodiment, thecancer is a cancer of the lung, thyroid, breast, ovary, pancreas,esophagus, kidney, liver, bladder, prostate, a melanoma, or ahematological malignancy. In an embodiment, the cancer is ahematological malignancy which is a lymphoma or a leukemia.

In an embodiment of the methods, the HHLA2 is a human HHLA2 protein. Inan embodiment of the methods, the HHLA2 protein comprises consecutiveamino acid residues having the sequence set forth in SEQ ID NO:1. In anembodiment of the methods, the antibody is a humanized antibody, achimeric antibody or an isolated human antibody. In an embodiment of themethods, the antibody is a monoclonal antibody.

Also provided is a method of treating an infectious disease a subject,comprising administering to the subject an amount of an anti-HHLA2antibody, or an HHLA2-binding fragment of an anti-HHLA2 antibody,effective to an infectious disease. In an embodiment of the methods, theHHLA2 is a human HHLA2 protein. In an embodiment of the methods, theHHLA2 protein comprises consecutive amino acid residues having thesequence set forth in SEQ ID NO:1. In an embodiment of the methods, theantibody is a humanized antibody, a chimeric antibody or an isolatedhuman antibody. In an embodiment of the methods, the antibody is amonoclonal antibody.

A method for identifying a biological sample as cancerous comprisingcontacting the sample with an anti-HHLA2 antibody, or antigen-bindingfragment thereof, having a detectable probe molecule attached theretoand determining if the anti-HHLA2 antibody, or antigen-binding fragmentthereof, having a detectable probe localizes to the sample by detectingthe probe.

In an embodiment, the probe is a fluorophore, a non-fluorescent dye, aradioactive isotope, a fluorescent protein, an enzyme, or a magnetic orparamagnetic entity. In an embodiment, the biological sample comprises atissue. In an embodiment, the biological sample comprises a cell.

In an embodiment, the biological sample is within a subject and theanti-HHLA2 antibody, or antigen-binding fragment thereof, having adetectable probe attached thereto is administered to the subject. In anembodiment, the biological sample has been obtained from a subject andthe sample is directly contacted with the anti-HHLA2 antibody, orantigen-binding fragment thereof, having a detectable probe attachedthereto. In an embodiment, the biological sample is a lung, thyroid,breast, ovary, pancreas, melanoma, esophagus, kidney, liver, bladder, orprostate sample. In an embodiment, the biological sample is determinedas cancerous if the HHLA2 antibody, or antigen-binding fragment thereof,having a detectable probe localizes to the sample.

In an embodiment, the biological sample is determined as cancerous ifthe amount of HHLA2 antibody, or antigen-binding fragment thereof,having a detectable probe localizing to the sample is greater than apredetermined control amount. The concept of a control, for examplecorresponding to an equivalent non-cancerous sample, is well-establishedin the art. In an embodiment, the biological sample is determined ascancerous if the sample mark's positive for HHLA2. In an embodiment, thesample is a sample taken from a tissue listed as positive for HHLA2expression in Table 3. In an embodiment, samples taken from a tissuelisted as negative for HHLA2 expression in Table 3 are excluded.

As used in the methods herein, the term “antibody” refers to an intactantibody, i.e. with complete Fc and Fv regions. “Fragment” refers to anyportion of an antibody, or portions of an antibody linked together, suchas, in non-limiting examples, a Fab, F(ab)₂, a single-chain Fv (scFv),which is less than the whole antibody but which is an antigen-bindingportion and which competes with the intact antibody of which it is afragment for specific binding. As such a fragment can be prepared, forexample, by cleaving an intact antibody or by recombinant means. Seegenerally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989), hereby incorporated by reference in its entirety).Antigen-binding fragments may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies or bymolecular biology techniques. In some embodiments, a fragment is an Fab,Fab′, F(ab′)₂, F_(d), F_(v), complementarity determining region (CDR)fragment, single-chain antibody (scFv), (a variable domain light chain(V_(L)) and a variable domain heavy chain (V_(H)) linked via a peptidelinker. In an embodiment the linker of the scFv is 10-25 amino acids inlength. In an embodiment the peptide linker comprises glycine, serineand/or threonine residues. For example, see Bird et al., Science, 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA,85:5879-5883 (1988) each of which are hereby incorporated by referencein their entirety), or a polypeptide that contains at least a portion ofan antibody that is sufficient to confer HHLA2-specific antigen bindingon the polypeptide, including a diabody. From N-terminus to C-terminus,both the mature light and heavy chain variable domains comprise theregions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol.196:901-917 (1987), or Chothia et al., Nature 342:878-883 (1989) , eachof which are hereby incorporated by reference in their entirety). Asused herein, the term “polypeptide” encompasses native or artificialproteins, protein fragments and polypeptide analogs of a proteinsequence. A polypeptide may be monomeric or polymeric. As used herein,an F_(d) fragment means an antibody fragment that consists of the V_(H)and CH1 domains; an F_(v) fragment consists of the V₁ and V_(H) domainsof a single arm of an antibody; and a dAb fragment (Ward et al., Nature341:544-546 (1989) hereby incorporated by reference in its entirety)consists of a V_(H) domain. In some embodiments, fragments are at least5, 6, 8 or 10 amino acids long. In other embodiments, the fragments areat least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150or 200 amino acids long.

The term “monoclonal antibody” as used herein refers to an antibodymember of a population of substantially homogeneous antibodies, i.e.,the individual antibodies comprising the population are identical exceptfor possible mutations, e.g., naturally occurring mutations, that may bepresent in minor amounts. Thus, the modifier “monoclonal” indicates thecharacter of the antibody as not being a mixture of discrete antibodies.In certain embodiments, such a monoclonal antibody typically includes anantibody comprising a polypeptide sequence that binds a target HHLA2 (oran HHLA2 receptor, as applicable), wherein the target-bindingpolypeptide sequence was obtained by a process that includes theselection of a single target binding polypeptide sequence from aplurality of polypeptide sequences. For example, the selection processcan be the selection of a unique clone from a plurality of clones, suchas a pool of hybridoma clones, phage clones, or recombinant DNA clones.In contrast to polyclonal antibody preparations, which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody of a monoclonal antibody preparation isdirected against a single determinant on an antigen. In addition totheir specificity, monoclonal antibody preparations are advantageous inthat they are typically uncontaminated by other immunoglobulins. Thus anidentified monoclonal antibody can be produced by non-hybridomatechniques, e.g. by appropriate recombinant means once the sequencethereof is identified.

In an embodiment of the inventions described herein, the antibody isisolated. As used herein, the term “isolated antibody” refers to anantibody that by virtue of its origin or source of derivation has one,two, three or four of the following: (1) is not associated withnaturally associated components that accompany it in its native state,(2) is free of other proteins from the same species, (3) is expressed bya cell from a different species, and (4) does not otherwise occur innature without the hand of man.

In an embodiment the composition or pharmaceutical compositioncomprising one or more of the antibodies or fragments described hereinis substantially pure with regard to the antibody or fragment. Acomposition or pharmaceutical composition comprising one or more of theantibodies or fragments described herein is “substantially pure” withregard to the antibody or fragment when at least about 60 to 75% of asample of the composition or pharmaceutical composition exhibits asingle species of the antibody or fragment. A substantially purecomposition or pharmaceutical composition comprising one or more of theantibodies or fragments described herein can comprise, in the portionthereof which is the antibody or fragment, 60%, 70%, 80% or 90% of theantibody or fragment of the single species, more usually about 95%, andpreferably over 99%. Antibody purity or homogeneity may tested by anumber of means well known in the art, such as polyacrylamide gelelectrophoresis or HPLC.

As used herein, a “human antibody” unless otherwise indicated is onewhose sequences correspond to (i.e. are identical in sequence to) anantibody that could be produced by a human and/or has been made usingany of the techniques for making human antibodies as disclosed herein,but not one which has been made in a human. This definition of a humanantibody specifically excludes a humanized antibody. A “human antibody”as used herein can be produced using various techniques known in theart, including phage-display libraries (e.g. Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991),hereby incorporated by reference in its entirety), by methods describedin Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,p. 77 (1985) (hereby incorporated by reference in its entirety); Boerneret al., J. Immunol., 147(1):86-95 (1991) (hereby incorporated byreference in its entirety), van Dijk and van de Winkel, Curr. Opin.Pharmacol., 5: 368-74 (2001) (hereby incorporated by reference in itsentirety), and by administering the antigen (e.g. HHLA2) to a transgenicanimal that has been modified to produce such antibodies in response toantigenic challenge, but whose endogenous loci have been disabled, e.g.,immunized xenomice (see, e.g., U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al. regardingXENOMOUSE™ technology, each of which patents are hereby incorporated byreference in their entirety), e.g. VelocImmune® (Regeneron, Tarrytown,N.Y.), e.g. UltiMab® platform (Medarex, now Bristol Myers Squibb,Princeton, N.J.). See also, for example, Li et al., Proc. Natl. Acad.Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated viaa human B-cell hybridoma technology. See also KM Mouse® system,described in PCT Publication WO 02/43478 by Ishida et al., in which themouse carries a human heavy chain transchromosome and a human lightchain transgene, and the TC mouse system, described in Tomizuka et al.(2000) Proc. Natl. Acad. Sci. USA 97:722-727, in which the mouse carriesboth a human heavy chain transchromosome and a human light chaintranschromosome, both of which are hereby incorporated by reference intheir entirety. In each of these systems, the transgenes and/ortranschromosomes carried by the mice comprise human immunoglobulinvariable and constant region sequences.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are sequences of human origin or identical thereto other thanantibodies naturally occurring in a human or made in a human.Furthermore, if the antibody (e.g. an intact antibody rather than, forexample, an Fab fragment) contains a constant region, the constantregion also is derived from such human sequences, e.g., human germlinesequences, or mutated versions of human germline sequences. The humanantibodies of the invention may include amino acid residues not encodedby human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. In one non-limiting embodiment, where the humanantibodies are human monoclonal antibodies, such antibodies can beproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

In an embodiment, the anti-HHLA2 antibody described herein is arecombinant human antibody. The term “recombinant human antibody”, asused herein, includes all human antibodies that are prepared, expressed,created or isolated by recombinant means, such as antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic or transchromosomalfor human immunoglobulin genes or a hybridoma prepared therefrom,antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, antibodies isolated from arecombinant, combinatorial human antibody library, and antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of all or a portion of a human immunoglobulin gene, sequencesto other DNA sequences. Such recombinant human antibodies have variableregions in which the framework and CDR regions are derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies can be subjected to in vitro mutagenesis(or, when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region (HVR) of the recipient are replaced by residuesfrom a HVR of a non-human species (donor antibody) such as mouse, rat,rabbit, or nonhuman primate having the desired specificity, affinity,and/or capacity. In some instances, FR residues of the humanimmunoglobulin variable domain are replaced by corresponding non-humanresidues. These modifications may be made to further refine antibodyperformance. Furthermore, in a specific embodiment, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. In an embodiment, the humanized antibodies do notcomprise residues that are not found in the recipient antibody or in thedonor antibody. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. See, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct.Biol. 2:593-596 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma &Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433(1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409, the contents of eachof which references and patents are hereby incorporated by reference intheir entirety. In one embodiment where the humanized antibodies docomprise residues that are not found in the recipient antibody or in thedonor antibody, the Fc regions of the antibodies are modified asdescribed in WO 99/58572, the content of which is hereby incorporated byreference in its entirety.

Techniques to humanize a monoclonal antibody are described in U.S. Pat.Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;5,693,762; 5,585,089; and 6,180,370, the content of each of which ishereby incorporated by reference in its entirety.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including antibodies having rodent or modified rodent V regions andtheir associated complementarity determining regions (CDRs) fused tohuman constant domains. See, for example, Winter et al. Nature 349:293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci. USA 86: 4220-4224(1989), Shaw et al. J. Immunol. 138: 4534-4538 (1987), and Brown et al.Cancer Res. 47: 3577-3583 (1987), the content of each of which is herebyincorporated by reference in its entirety. Other references describerodent hypervariable regions or CDRs grafted into a human supportingframework region (FR) prior to fusion with an appropriate human antibodyconstant domain. See, for example, Riechmann et al. Nature 332: 323-327(1988), Verhoeyen et al. Science 239: 1534-1536 (1988), and Jones et al.Nature 321: 522-525 (1986), the content of each of which is herebyincorporated by reference in its entirety. Another reference describesrodent CDRs supported by recombinantly veneered rodent frameworkregions - European Patent Publication No. 0519596 (incorporated byreference in its entirety). These “humanized” molecules are designed tominimize unwanted immunological response toward rodent anti-humanantibody molecules which limits the duration and effectiveness oftherapeutic applications of those moieties in human recipients. Theantibody constant region can be engineered such that it isimmunologically inert (e.g., does not trigger complement lysis). See,e.g. PCT Publication No. WO99/58572; UK Patent Application No.9809951.8. Other methods of humanizing antibodies that may also beutilized are disclosed by Daugherty et al., Nucl. Acids Res. 19:2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867;5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO01/27160 (each incorporated by reference in their entirety).

Other forms of humanized antibodies have one or more CDRs (CDR L1, CDRL2, CDR L3, CDR H1, CDR H2, or CDR H3) which are altered with respect tothe original antibody, which are also termed one or more CDRs “derivedfrom” one or more CDRs from the original antibody.

In embodiments, the antibodies or fragments herein can be producedrecombinantly, for example antibodies expressed using a recombinantexpression vector transfected into a host cell, antibodies isolated froma recombinant, combinatorial human antibody library, antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes.

In an embodiment, the anti-HHLA2 antibody described herein (or fragmentthereof) is capable of specifically binding HHLA2 or specifically bindsan HHLA2. IN an embodiment, the HHLA2 is human a HHLA 2. As used herein,the terms “is capable of specifically binding” or “specifically binds”refers to the property of an antibody or fragment of binding to the(specified) antigen with a dissociation constant that is <1 μM,preferably <1 nM and most preferably <10 pM. In an embodiment, the Kd ofthe antibody (or fragment) for HHLA2 is 250-500 pM. An epitope that“specifically binds” to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecular entityis said to exhibit “specific binding” or “preferential binding” if itreacts or associates more frequently, more rapidly, with greaterduration and/or with greater affinity with a particular cell orsubstance than it does with alternative cells or substances. An antibody“specifically binds” or “preferentially binds” to a target if it bindswith greater affinity, avidity, more readily, and/or with greaterduration than it binds to other substances. For example, an antibodythat specifically or preferentially binds to a HHLA2 conformationalepitope is an antibody that binds this epitope with greater affinity,avidity, more readily, and/or with greater duration than it binds toother HHLA2 epitopes or non-HHLA2 epitopes. It is also understood byreading this definition that, for example, an antibody (or moiety orepitope) that specifically or preferentially binds to a first target mayor may not specifically or preferentially bind to a second target. Assuch, “specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding.

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding portion thereof, binds toan epitope in a manner sufficiently similar to the binding of a secondantibody, or an antigen-binding portion thereof, such that the result ofbinding of the first antibody with its cognate epitope is detectablydecreased in the presence of the second antibody compared to the bindingof the first antibody in the absence of the second antibody. Thealternative, where the binding of the second antibody to its epitope isalso detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate, based upon the teachings provided herein, that suchcompeting and/or cross-competing antibodies are encompassed and can beuseful for the methods disclosed herein.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. The antibody or fragment can be, e.g., any of an IgG, IgD, IgE,IgA or IgM antibody or fragment thereof, respectively. In an embodimentthe antibody is an immunoglobulin G. In an embodiment the antibodyfragment is a fragment of an immunoglobulin G. In an embodiment theantibody is an IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4. In an embodimentthe antibody comprises sequences from a human IgG1, human IgG2, humanIgG2a, human IgG2b, human IgG3 or human IgG4. A combination of any ofthese antibodies subtypes can also be used. One consideration inselecting the type of antibody to be used is the desired serum half-lifeof the antibody. For example, an IgG generally has a serum half-life of23 days, IgA 6 days, IgM 5 days, IgD 3 days, and IgE 2 days. (Abbas A K,Lichtman A H, Pober J S. Cellular and Molecular Immunology, 4th edition,W.B. Saunders Co., Philadelphia, 2000, hereby incorporated by referencein its entirety).

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “V_(H).” Thevariable domain of the light chain may be referred to as “V_(L).” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites. The term “variable” refers to the fact thatcertain portions of the variable domains differ extensively in sequenceamong antibodies and are used in the binding and specificity of eachparticular antibody for its particular antigen. However, the variabilityis not evenly distributed throughout the variable domains of antibodies.It is concentrated in three segments called hypervariable regions (HVRs)both in the light-chain and the heavy-chain variable domains. The morehighly conserved portions of variable domains are called the frameworkregions (FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “hypervariable region” or “HVR” when used herein refers to theregions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the V_(H) (H1, H2, H3) and three in theV_(L) (L1, L2, L3). In native antibodies, H3 and L3 display the mostdiversity of the six HVRs, and H3 in particular is believed to play aunique role in conferring fine specificity to antibodies. See, e.g., Xuet al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods inMolecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).Indeed, naturally occurring camelid antibodies consisting of a heavychain only are functional and stable in the absence of light chain. See,e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff etal., Nature Struct. Biol. 3:733-736 (1996). A number of HVR delineationsare in use and are encompassed herein. The Kabat ComplementarityDetermining Regions (CDRs) are based on sequence variability and are themost commonly used (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991) hereby incorporated by reference in its entirety).Chothia refers instead to the location of the structural loops (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent acompromise between the Kabat HVRs and Chothia structural loops, and areused by Oxford Molecular's AbM antibody modeling software. The “contact”HVRs are based on an analysis of the available complex crystalstructures. HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35(H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH.The variable domain residues are numbered according to Kabat et al.,supra, for each of these definitions.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine of the Fc region may be removed, for example, duringproduction or purification of the antibody, or by recombinantlyengineering the nucleic acid encoding a heavy chain of the antibody.Accordingly, an intact antibody as used herein may be an antibody withor without the otherwise C-terminal cysteine.

Compositions or pharmaceutical compositions comprising the antibodies,ScFvs or fragments of antibodies disclosed herein are preferablycomprise stabilizers to prevent loss of activity or structural integrityof the protein due to the effects of denaturation, oxidation oraggregation over a period of time during storage and transportationprior to use. The compositions or pharmaceutical compositions cancomprise one or more of any combination of salts, surfactants, pH andtonicity agents such as sugars can contribute to overcoming aggregationproblems. Where a composition or pharmaceutical composition of thepresent invention is used as an injection, it is desirable to have a pHvalue in an approximately neutral pH range, it is also advantageous tominimize surfactant levels to avoid bubbles in the formulation which aredetrimental for injection into subjects. In an embodiment, thecomposition or pharmaceutical composition is in liquid form and stablysupports high concentrations of bioactive antibody in solution and issuitable for parenteral administration, including intravenous,intramuscular, intraperitoneal, intradermal and/or subcutaneousinjection. In an embodiment, the composition or pharmaceuticalcomposition is in liquid form and has minimized risk of bubble formationand anaphylactoid side effects. In an embodiment, the composition orpharmaceutical composition is isotonic. In an embodiment, thecomposition or pharmaceutical composition has a pH or 6.8 to 7.4.

In an embodiment the ScFvs or fragments of antibodies disclosed hereinare lyophilized and/or freeze dried and are reconstituted for use.

Examples of pharmaceutically acceptable carriers include, but are notlimited to, phosphate buffered saline solution, sterile water (includingwater for injection USP), emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline, for example 0.9% sodium chloride solution, USP. Compositionscomprising such carriers are formulated by well known conventionalmethods (see, for example, Remington's Pharmaceutical Sciences, 18thedition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; andRemington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000, the content of each of which is hereby incorporated inits entirety). In non-limiting examples, the can comprise one or more ofdibasic sodium phosphate, potassium chloride, monobasic potassiumphosphate, polysorbate 80 (e.g.2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl(E)-octadec-9-enoate), disodium edetate dehydrate, sucrose, monobasicsodium phosphate monohydrate, and dibasic sodium phosphate dihydrate.

The antibodies, or fragments of antibodies, or compositions, orpharmaceutical compositions described herein can also be lyophilized orprovided in any suitable forms including, but not limited to, injectablesolutions or inhalable solutions, gel forms and tablet forms.

The term “Kd”, as used herein, is intended to refer to the dissociationconstant of an antibody-antigen interaction. One way of determining theKd or binding affinity of antibodies to HHLA2 is by measuring bindingaffinity of monofunctional Fab fragments of the antibody. (The affinityconstant is the inverted dissociation constant). To obtainmonofunctional Fab fragments, an antibody (for example, IgG) can becleaved with papain or expressed recombinantly. The affinity of ananti-HHLA2 Fab fragment of an antibody can be determined by surfaceplasmon resonance (BIAcore3000TM surface plasmon resonance (SPR) system,BIAcore Inc., Piscataway N.J.). CM5 chips can be activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NETS) according to the supplier's instructions.HHLA2 can be diluted into 10 mM sodium acetate pH 4.0 and injected overthe activated chip at a concentration of 0.005 mg/mL. Using variableflow time across the individual chip channels, two ranges of antigendensity can be achieved: 100-200 response units (RU) for detailedkinetic studies and 500-600 RU for screening assays. Serial dilutions(0.1-10× estimated Kd) of purified Fab samples are injected for 1 min at100 microliters/min and dissociation times of up to 2 h are allowed. Theconcentrations of the Fab proteins are determined by ELISA and/orSDS-PAGE electrophoresis using a Fab of known concentration (asdetermined by amino acid analysis) as a standard. Kinetic associationrates (k_(on)) and dissociation rates (k_(off)) are obtainedsimultaneously by fitting the data to a 1:1 Langmuir binding model(Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110, the content of which is hereby incorporated in itsentirety) using the BIA evaluation program. Equilibrium dissociationconstant (Kd) values are calculated as k_(off)/k_(on). This protocol issuitable for use in determining binding affinity of an antibody orfragment to any HHLA2. Other protocols known in the art may also beused. For example, ELISA of HHLA2 with mAb can be used to determine thekD values. The Kd values reported herein used this ELISA-based protocol.

Also provided is a composition comprising an HHLA2-immunoglobulin-fusionprotein bound to an HHLA2 receptor on an immune system cell. In anembodiment, the HHLA2-immunoglobulin-fusion protein comprises theextracellular domain of human HHLA2. In an embodiment, theHHLA2-immunoglobulin-fusion protein comprises a human IgG Fc region. Inan embodiment, the IgG is an IgG1. In an embodiment, the immune systemcell is a CD4 T cell, an CD8 T cell, or an antigen-presenting cell. Inan embodiment, the immune system cell is human.

Also provided is a composition comprising an HHLA2-immunoglobulin-fusionprotein and a pharmaceutically acceptable carrier. In an embodiment,HHLA2-immunoglobulin-fusion protein comprises an extracellular domain ofhuman HHLA2. In an embodiment, HHLA2-immunoglobulin-fusion proteincomprises a human IgG Fc region. In an embodiment, the IgG is an IgG1.In an embodiment, the HHLA2-immunoglobulin-fusion protein comprises theconsecutive amino acid residues set forth in SEQ ID NO:2. In anembodiment, the composition is a pharmaceutical composition.

Also provided is a composition comprising an anti-HHLA2 antibody, or anHHLA2-binding fragment of such an antibody, and a pharmaceuticallyacceptable carrier. In an embodiment, the HHLA2 is a human HHLA2protein. In an embodiment, the HHLA2 protein comprises consecutive aminoacid residues having the sequence set forth in SEQ ID NO:1. In anembodiment, the antibody is a humanized antibody, a chimeric antibody oran isolated human antibody. In an embodiment, the antibody is amonoclonal antibody. In an embodiment, the composition is apharmaceutical composition.

Also provided is an HHLA2-immunoglobulin-fusion protein for treating anautoimmune disease in a subject, or for suppressing transplant rejectionin a subject.

Also provided is an anti-HHLA2 antibody, or an HHLA2-binding fragment ofsuch an antibody, for treating a cancer in a subject.

Also provided is a screening method for identifying an agent as atreatment for an autoimmune disease in a subject, or as a treatment forsuppressing transplant rejection in a subject, comprising contacting animmune system cell expressing an HHLA2 receptor with the agent andquantifying activity of the cell in the presence and the absence of theagent, and identifying the agent as a treatment for an autoimmunedisease in a subject, or as a treatment for suppressing transplantrejection in a subject,

wherein an agent that inhibits the activity of the cell in its presenceas compared to in its absence is indicated to be a treatment for anautoimmune disease in a subject or a treatment for suppressingtransplant rejection in a subject, and an agent that does not affectactivity of, or stimulates activity of , the cell its presence ascompared to in its absence is not indicated to be a treatment for anautoimmune disease in a subject or a treatment for suppressingtransplant rejection in a subject.

Also provided is a screening method for identifying an agent as atreatment for a cancer or for an infectious disease, comprisingcontacting an HHLA2 receptor with the agent and an amount of HHLA2 andquantifying the antagonism of the agent on HHLA2 binding to the HHLA2receptor, and identifying the agent as a treatment for a cancer or foran infectious disease,

wherein an agent that competes with, and/or reduces, HHLA2 binding tothe HHLA2 receptor in its presence as compared to in its absence isindicated to be a treatment for a cancer or an infectious disease, andan agent that does not affect HHLA2 binding in its presence as comparedto in its absence is not indicated to be a treatment for a cancer or foran infectious disease. In an embodiment, the HHLA2 receptor is expressedon a cell. In an embodiment, the cell is an immune system cell. In anembodiment, the cell is CD4 T cell, a CD9 T cell or anantigen-presenting cell.

In an embodiment of the screening methods, the agent is an organicmolecule of 2000 daltons or less, an antibody, an antigen-bindingfragment of an antibody, and siRNA nucleic acid, a polypeptide of lessthan 200 residues or an aptamer.

In an embodiment, “determining” as used herein means experimentallydetermining.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

Experimental Details

Introduction

Here the HERV-HLTR-associating 2 (HHLA2) (29) is described as a memberof the B7 family with coinhibitory function for both human CD4 and CD8 Tcells, which is comparable to other important family members. A putativereceptor for HHLA2 is expressed widely on T cells and APCs.

Results

Characterization of HHLA2 as a B7 family member: In a homology search ofvarious databases using amino acid sequences of human B7x and B7-H3,HHLA2 was identified which was shown previously to share significanthomology with the B7 family (29, 30) and was also called B7H7(30). Thehuman HHLA2 gene is located in the q13.13 region of the chromosome 3 andis near the B7-1 and B7-2 genes (q13.3-q21). The open reading frame wassequenced and the deduced protein sequence of HHLA2 was found to contain414 amino acids (FIG. 1A), longer than most B7 members, but shorter thanhuman B7-H3. HHLA2 shares varying levels of amino acid identity andsimilarity with human B7-1 (10% and 23%), B7-2 (13% and 29%), B7h (15%and 30%), PD-L1 (12% and 26%), PD-L2 (14% and 27%), B7-H3 long form (15%and 31%) and short form (16% and 33%), and B7x (18% and 30%), which arecomparable to the homologies exhibited by other members of the family;for example, B7-1, the founding member of the B7 family, shares 13-21%of amino acid identity and 22-37% of similarity with other human B7molecules.

The putative HHLA2 protein has an N-terminal signal peptide, anectodomain composed of tandem IgV-IgC-IgV domains, six potential sitesfor N-linked glycosylation, a transmembrane region, and a 49-aacytoplasmic tail (FIG. 1A). The predicted HHLA2 protein is a type Itransmembrane molecule. To test this prediction, the HHLA2 proteinlocalization was examined by expressing HHLA2-YFP fusion protein in the3T3 cell line which did not express endogenous HHLA2. Confocalmicroscopy analysis revealed that HHLA2 protein was predominantly foundon cell membranes with some in the cytoplasm (FIG. 1B). In contrast,human CTLA-4-YFP fusion protein was mainly localized intracellularly inthe 3T3 cell (FIG. 1B).

Evolution of HHLA2: In a previous study the B7 family of proteins wasdivided into three groups by phylogenetic analysis (23). With HHLA2added into this family, PAUP 4.0b10 (31) was used to reevaluate therelationship among human B7 proteins. As shown in FIG. 1C, aphylogenetic comparison of the family divided the human B7 moleculesinto three groups: group I includes B7-1, B7-2, and B7h; group IIconsists of PD-L1 and PD-L2; and group III contains B7x, HHLA2 andB7-H3. For group I, CD28 and CTLA-4 are receptors for all three B7molecules and the closely related ICOS is a receptor for B7h, whereasPD-L1 can bind B7-1. For group II, PD-1 is the receptor for both PD-L1and PD-L2. For group III, receptors have not been identified yet. Thephylogenetic comparison suggests that receptors for group III would notbe real homologues of receptors for group I and II.

Based on sequence analyses, putative HHLA2 orthologs appear to bepresent in wide range of species, including fish (GeneBank accessionnumber ACH85300), frog (NP_001122116), Heterocephalus glaber (EHB18400),giant panda (EFB27984), and monkey (EHH16036 and EHH51009), suggestingevolutionally conserved function. However, in contrast to other B7family members, laboratory mouse and rat strains do not express HHLA2,which makes it the first B7 family member expressed in human but not inmouse.

Protein expression pattern of HHLA2: The expression of HHLA2 at theprotein level is completely unknown at present. To examine the proteinexpression, a panel of monoclonal antibodies (mAbs) was generatedagainst HHLA2 from mice as mice do not express the HHLA2 gene. Thespecificity of the mAbs was demonstrated by ELISA and FACS in which mAbsreacted with HHLA2 but not with other B7 molecules (FIG. 2A).

Using the anti-HHLA2 mAb clone 566.1, HHLA2 expression was examined onAPCs by FACS. CD14 positive monocytes in human peripheral bloodmononuclear cells (PBMCs) expressed significant levels of HHLA2 and theexpression was further up-regulated by stimulation with LPS and IFN-γ(FIG. 2B). Resting CD19 positive B cells did not express HHLA2, but theexpression was induced by LPS and IFN-γ stimulation (FIG. 2B). No clearHHLA2 protein could be detected on blood monocyte-derived immaturedendritic cells (DCs) or LPS/IFN-γ-induced mature DCs (FIG. 2B). For Tcells, both CD4 and CD8 T cells in PBMCs did not express HHLA2; bothwere still negative after stimulation with plate-bound anti-CD3 (FIG.2B). As a control, PD-L1, another B7 molecule, was induced on monocytes,B cells and DCs after LPS/IFN-y stimulation and was induced on CD4 andCD8 T cells after stimulation with anti-CD3 (FIG. 2B). Collectively,these results demonstrate that endogenous HHLA2 is an integral cellsurface protein constitutively expressed on monocytes and induced on Bcells.

HHLA2 does not bind other known members of the CD28 and B7 families: Allpreviously characterized B7 family members can act as ligands andregulate T cell function by binding to receptors. The receptor for HHLA2is unknown, therefore it was first asked whether any of the known CD28family members are the receptor for HHLA2. To this end, an HHLA2-Igfusion protein was generated consisting of the extracellular portion ofhuman HHLA2 and the Fc portion of IgG1 and three other controlsincluding B7x-Ig, B7-H3-Ig, and only the Fc portion of human IgG1 (Igcontrol). The HHLA2-Ig fusion protein and the controls, produced in thesame system and purified in the same way, were used to search for theinteractions between HHLA2 and the CD28 family members using FACSanalysis. To do this 3T3 lines expressing cell surface human CD28, ICOS,PD-1 (FIG. 3A) were established. CTLA-4 is not primarily a cell surfaceprotein (FIG. 1B), mainly due to the fact that it contains anintracellular localization motif (TTGVYVKMPPT) (SEQ ID NO:3) in itscytoplasmic tail (32). A 3T3 line expressing cell surface CTLA-4 wasestablished which did not contain the cytoplasmic tail (FIG. 3A). InFACS experiments, HHLA2-Ig, like the other control Igs (Ig, B7x-Ig andB7-H3-Ig), did not bind CD28, CTLA-4, ICOS, and PD-1 on the cell surfaceof 3T3 cells (FIG. 3B). Within the B7 family, B7-1 can bind to anotherB7 molecule PD-L1 (21). To test whether HHLA2 binds to any of known B7family members, 3T3 and CT26 lines were established that expressed cellsurface human B7-1, B7-2, B7h, PD-L1, PD-L2, B7-H3 and B7x, and foundthat neither HHLA2-Ig nor B7x-Ig bound the other B7 molecules (FIG. 3B).As a positive control, PD-1-Ig bound 3T3 cells expressing PD-L1 or PD-L2(FIG. 3C). These results reveal that neither the known members of theCD28 family nor those of the B7 family interact with HHLA2.

Constitutive expression of an HHLA2 putative receptor on T cells andother immune cells. T cells express receptors for members of the B7family. To test whether T cells have an HHLA2 receptor, HHLA2-Ig wasused and control Ig to stain T cells from PBMCs. FACS analyses showedthat HHLA2 bound freshly isolated resting CD4 and CD8 T cells (FIG. 4A).After stimulation with plate-bound anti-CD3 for three days, activatedCD4 and CD8 T cells still expressed a receptor for HHLA2 (FIG. 4A). Incontrast, ICOS was not expressed on resting CD4 and CD8 T cells but wasinduced after stimulation (FIG. 4A). HHLA2 receptor positive cells andICOS positive cells were partially overlapping. PD-1, another CD28family member, was recently reported to be expressed on human B cells(33). It was therefore examined whether APCs have a receptor for HHLA2.HHLA2-Ig bound freshly isolated B cells and monocytes, suggesting thesecells express a receptor for HHLA2 (FIG. 4A). PBMCs were furtherstimulated with LPS/IFN-γ for three days, and it was found B cells andmonocytes were activated, as evidenced by induced expression of PD-L1.Both activated B cells and monocytes were stained by HHLA2-Ig (FIG. 4A),suggesting resting and activated B cells as well as monocytes have aputative HHLA receptor. Finally, dendritic cells (DCs) were examined. Itwas found that HHLA2 bound blood monocyte-derived immature DCs as wellas LSP/IFN-γ-induced mature DCs (FIG. 4A). In contrast to immune cells,HHLA2 did not bind human HeLa cells and mouse 3T3 cells (FIG. 4B),suggesting these cells did not have an HHLA2 receptor. Taken together,these results indicate that a putative HHLA2 receptor is constitutivelyexpressed on T cells, B cells, monocytes, and DCs.

HHLA2 inhibits TCR-mediated CD4 and CD8 T cell proliferation: Based onthe data showing that HHLA2 protein was detected on APCs and a putativereceptor was constitutively expressed on T cells, it was examinedwhether HHLA2 was able to regulate T cell function using a systemmodified from previous studies (23). In this system, purified T cellswere activated with plate-bound mAb to human CD3 and the activation of Tcells was determined on day three and five. Firstly a dose titration wasperformed of anti-CD3 and it was found that T cells from differentnormal donors needed different concentrations of anti-CD3 to achieve themiddle level of proliferation. Therefore, different suitableconcentrations of anti-CD3 were used for T cell experiments. The MTTassay was used to quantify anti-CD3 induced T cell activation in thepresence of immobilized HHLA2-Ig, control Ig, or B7x-Ig (FIG. 5A).HHLA2-Ig significantly decreased T cell activation induced by anti-CD3(FIG. 5A). As a control, B7x-Ig also inhibited T cell activation in thesame system (23). As both CD4 and CD8 T cells constitutively express anHHLA2 receptor, it was next examined whether HHLA2 was able to inhibitboth CD4 and CD8 T cells. Purified T cells from PBMCs were labeled withcarboxyfluorescein diacetate succinimidyl ester (CFSE), and stimulatedwith anti-CD3 in the presence of immobilized HHLA2-Ig, control Ig, orB7x-Ig for five days. These cells were then analyzed by FACS withanti-CD4 and anti-CD8. HHLA2-mediated inhibition was determined bygating on CD4 and CD8 T cell populations and measuring CFSE fluorescenceintensity. Both CD4 and CD8 T cells proliferated vigorously whenincubated with anti-CD3 and control Ig (FIG. 5B,C), with more than 55%of CD4 and 69% of CD8 T cells dividing. However, when T cells wereincubated with anti-CD3 and HHLA2-Ig, significantly fewer CD4 and CD8 Tcells proliferated, with less than 38% of CD4 and 52% of CD8 T cellsdividing (FIG. 5B,C). Similarly, B7x-Ig also inhibited both CD4 and CD8T cell proliferation, with less than 44% of CD4 and 57% of CD8 T cellsdividing (FIG. 5 ). These findings from two functional assaysdemonstrate that HHLA2 inhibits TCR-mediated proliferation of both humanCD4 and CD8 T cells.

HHLA2 inhibits cytokine production from T cells: Next tested was theeffect of HHLA2 on cytokine production from T cells. Purified T cellsfrom PBMCs were stimulated with anti-CD3 in the presence of immobilizedHHLA2-Ig or control Ig for three days and cytokines in the supernatantswere measured using Th1/Th2/Th9/Th17/Th22 flowcytomix. Among the 13 Tcell-derived cytokines tested, it was found HHLA2 significantly reducedproduction of 7 cytokines from T cells: IFN-γ (21% reduction), TNF-α(30% reduction), IL-5 (39% reduction), IL-10 (56% reduction), IL-13 (39%reduction), IL-17A (36% reduction), and IL-22 (35% reduction) (FIG. 6 ).HHLA2 reduced production of IL-2 and IL-9, but the differences did notreach statistical significance. In addition, there was no effect ofHHLA2 on cytokine production of IL-1β, IL-4, IL-6, and IL-12p70. Theseresults suggest that HHLA2 is able to suppress certain cytokinesproduced from T cells induced by TCR signaling.

HHLA2 protein has limited expression in normal tissues: Using monoclonalantibodies to HHLA, an immunohistochemistry (IHC) protocol was developedto evaluate HHLA2 protein expression in normal human organs and commoncancers. It was found that HHLA2 protein was in the epithelium of thegut, breast, placenta, and gallbladder, but not in other organs such asskin, liver, uterine cervix, subcutis, prostate, pancreas, spleen,tonsils, umbilical cord, lymph node, esophagus, adrenal, ovary, stomach,thyroid, lung, thymus, larynx, brain, aorta, uterus, and salivary glands(Table 1).

HHLA2 protein is over-expressed in many human cancers: Using IHC, it wasfound that HHLA2 was over-expressed in 50% or more of the cancerouslung, thyroid, breast, ovary, pancreas, and melanoma samples (Table 2and FIG. 7 ). HHLA2 was also expressed in other cancerous esophagus,kidney, liver, bladder, and prostate (Table 2 and FIG. 7 ). In addition,some hematological malignancies were found including lymphoma andleukemia expressed HHLA2 on their surface by flow cytometry (Table 3).

TABLE 1 IHC staining for HHLA2 protein in normal human organs. NormalTissues (Number Positive/Total Cores Analyzed) Skin (0/2) Liver (0/2)Uterine Cervix (0/4) Subcutis (0/2) Gallbladder (5/11) Prostate (0/2)Breast (2/2) Pancreas (0/2) Placenta (2/2) Spleen (0/2) Tonsils (0/2)Umbilical Cord (0/2) Lymph Node (0/12) Esophagus (0/2) Adrenal (0/2)Ovary (0/1) Stomach (0/2) Thyroid (0/2) Lung (0/2) Small Intestine (2/2)Thymus (0/2) Larynx (0/9) Colon (2/2) Brain (0/6) Aorta (0/2) Kidney(3/4) Uterus (0/9) Salivary Glands (0/2)

TABLE 2 IHC staining for HHLA2 protein in human common cancers fromvarious organs Cancer Samples (Number Positive/Total Cores) Stomach(0/10) Breast (7/10) Endometrial (0/9) Esophagus(2/10) Liver (4/10)Gallbladder (0/10) Lung (6/9) Bladder (4/10) Larynx (0/10) Colon/Rectum(3/8) Ovary (4/8) Uterine Cervix (0/10) Thyroid (6/9) Pancreas (5/10)Lymphoma (0/10) Kidney (2/6) Prostate (3/9) Malignant Melanoma (5/9)

TABLE 3 Expression of HHLA2 protein in various human cancer cell linesdetermined by flow cytometry. HHLA2 Human Cell Lines Derived FromExpression U937 Histiocytic Lymphoma + Raji Burkitt lymphoma + HL60Acute Promyelocytic Leukemia + MOLM13 Acute monocytic leukemia + K562Chronic myelogenous leukemia + CEM Acute lymphoblastic leukemia +SK-BR-5 Breast carcinoma (Metastasis) + HCC-1143 Breast ductalcarcinoma + MDA-MB-231 Breast adenocarcinoma + BT-20 Breast carcinoma +BT-549 Breast ductal carcinoma + SK-BR-7 Breast carcinoma − SK-LC-1 Lungadenocarcinoma − SK-LC-7 Lung adenocarcinoma − SK-OV-6 Ovarian carcinoma− Hela Cervical cancer − Jurkat Acute T cell leukemia − THP-1 Acutemonocytic leukemia − KG1 Acute myelogenous leukemia −

Discussion

Herein is provided evidence for HHLA2 as a new member of the B7 familythat inhibits proliferation and cytokine production of both human CD4and CD8 T cells. This is significant and unexpected in light of previousreports of methods of using pHHLA2 to co-stimulate T-cells (see USPatent Application Publication 20090175876). HHLA2 was originally clonedas a gene that was polyadenylated within a long terminal repeat (LTR) ofthe HERV-H endogenous retrovirus family (29), exhibiting homology withB7 (29, 30). It was found that HHLA2 has all the characteristics of a B7family member. Similar to other members of the B7 family, HHLA2 shares10-18% of amino acid identity and 23-33% of similarity to other human B7molecules. It is already demonstrated that the IgV domain is thereceptor-binding domain for B7-1 (34), B7-2 (35), PD-L1 (36) and PD-L2(37). Like other B7s, HHLA2 has extracellular IgV and IgC domains. Withthe phylogenetic analyses, it was found that HHLA2 formed the thirdgroup with B7x and B7-H3 within the B7 family. Indeed, the highesthomology sequences to HHLA2 are B7-H3 and B7x. The bioinformaticanalyses and other results (29, 30) reveal that HHLA2 is found invarious species including human, monkey, frog, and fish, but is notexpressed in mouse and rat. Mouse and rat have only HHLA2 pseudogenes(30). This is unique as all other known members of the B7 family and ofthe CD28 family are found in both human and mouse.

Compared to other B7s, human HHLA2 has a different expression pattern.HHLA2 protein was expressed highly and constitutively on monocytes,while its expression on human B cells was induced by inflammatorystimulation. However, blood monocyte-derived DCs and T cells were HHLA2negative even after activation with LPS/IFN-γ and anti-CD3,respectively. Differently from HHLA2, it was found that the other B7molecule, PD-L1, was not expressed in human resting T cells and APCs butwas induced on these immune cells after activation. B7-2 is expressed atvery low levels on human resting B cells and immature DCs and is inducedto high levels with stimuli (38, 39), while B7x is hardly detected onnormal human T cells and APCs (38). These studies highlight the dramaticdifferences in the spatial and temporal expression of the individualmembers of the B7 family.

HHLA2 appears to have a counter-receptor that is distinct from CD28,CTLA-4, ICOS, PD-1 and all B7 molecules. CD28 is constitutivelyexpressed on T cells, whereas CTLA-4, ICOS, and PD-1 are induced after Tcell activation (1-3). HHLA2-Ig fusion protein did not interact with anyknown members of the CD28 and B7 families, demonstrating that thesemolecules are not the receptor for HHLA2. These results are consistentwith the phylogenetic analyses which suggests the receptors for thegroup III (HHLA2, B7x, and B7-H3) may be distinct from the receptors forgroups I and II. Interestingly, HHLA2 bound not only to activated humanCD4 and CD8 T cells but also to resting CD4 and CD8 T cells. Therefore,HHLA2 joins B7-1 and B7-2 to recognize receptors expressed on bothresting and activated T cells.

HHLA2 is able to function as a negative regulator of human T cells: Inthe presence of TCR signaling, immobilized HHLA2 protein suppressedproliferation of both human CD4 and CD8 T cells as effectively as B7x inthe same experimental system. The second line of evidence that supportedan inhibitory role for HHLA2 in T cell regulation is its effect oncytokine production. Among 13 cytokines from T cells induced by TCRsignaling, HHLA2 significantly reduced the production of 7 cytokinesincluding IFN-γ, TNF-α, IL-5, IL-10, IL-13, IL-17A, and IL-22,indicating that HHLA2 is able to inhibit T cell cytokine production. Itwas found that human T cells from different donors had considerablevariation in cytokine production, which may reflect an interestingheterogeneity in the human response. In the group III of the B7 family,B7x suppresses T cells and is widely over-expressed in many human solidtumors; B7-H3 is reported to have costimulatory and coinhibitory effects(26), although clinical observations suggest that it functions in tumorimmune evasion. This group is now expanded to include HHLA2 as a T cellcoinhibitor. In addition to T cells, human APCs also express a receptorfor HHLA2.

In summary, herein is characterized a member of the B7 family thatserves as an attenuator of T cell responses. It is the first B7 familymember identified to exist in humans but not in mice. Its putativereceptor is constitutively expressed on human T cells and APCs. Theexpression patterns of HHLA2 and its putative counter-receptor coupledwith its coinhibitory function suggests that this pathway is a potentregulator of human immune responses at both the very early and latestages. Notably, in the clinic, CTLA-4-Ig fusion proteins (Abatacept andBelatacept) inhibit T cell functions and have already been used to treatadult rheumatoid arthritis and to prevent acute kidney transplantrejection (40, 41), respectively, a mAb blocking CTLA-4 functions(Ipilimumab) was recently approved for treatment of metastatic melanoma(42, 43) and some mAbs against PD-1 and PD-L1 are currently in clinicaltrials with cancer patients (44, 45). Similarly, the newly-identifiedinhibitory HHLA2 pathway permits new therapies for human cancers,autoimmune disorders, infection, and transplant rejection.

The HHLA2 expression results in normal and cancerous tissues indicatethat targeting HHLA2 can be a way to deliver drugs and antibodies tocancers. For example, molecules which can bind HHLA2 protein, such asanti-HHLA2 antibody or antibody-drug conjugate (ADC) and HHLA2 receptorsor receptor-drug conjugates, can be used to treat human cancers whichexpress HHLA2. In addition, HHLA2 expression can be used as a diagnosticmarker for many cancers. Because most normal tissues do not expressHHLA2, but most human cancer do express HHLA2, then HHLA2 expression canbe used as a diagnostic marker for many cancers.

Materials and Methods

Bioinformatic analysis. BLAST was used to search public databases withprotein sequences. Sequence alignment and homology comparison were donewith MacVector 10.6. The phylogenetic tree was generated by PAUP(4.0b10) using sequence alignment by removal of significant inserts andtrimming C- and N-terminal extensions (31). Motifs and domains wereanalyzed with EMBL-EBI tools, SMART, and CBS Prediction.

Mice and cells. BALB/c mice were purchased from the National CancerInstitute and maintained under specific pathogen-free conditions. HumanPBMCs were isolated by Ficoll-Hypaque gradient centrifugation. Allprotocols were reviewed and approved by the Albert Einstein College ofMedicine Institutional Animal Care and Use Committee and InstitutionalReview Board. Cell lines were cultured in complete DMEM or RPMI1640media.

Production and purification of fusion proteins. HHLA2-Ig and B7x-Igproteins were prepared by fusing the coding region of the extracellulardomain without signal peptide of human HHLA2 or B7x to a human IgG1 Fctag of plasmid pMT/BiP V2 as described (23). The pMT/BiP V2 constructitself produced human IgG1 Fc tag as a control. All constructs wereco-transfected into Drosophila cell line S2 with a hygromycin resistanceplasmid, and the stable transfected cell lines were induced to secretefusion proteins in Express Five serum-free medium (LifeTechnologies).Proteins were purified on Protein G Plus Agarose columns (Pierce) andthen FPLC. The purity and identity of fusion proteins were confirmed bySDS-PAGE, Western blotting, and protein sequencing with MALDI-TOF-MS/MS.

Retrovirus constructs and cell line transfectants. HHLA2-YFP fusionprotein construct was generated by using PCR to amplify the codingsequence of HHLA2 without the stop codon and then cloned into the Bgl IIsite of the L50-YFP/MSCV vector. CTLA-4 in L50-FYP/MSCV vector wasreported previously (46). The coding sequences of human CD28, PD-1,ICOS, B7-1, B7-2, B7h, PD-L1, PD-L2, B7-H3, and B7x were cloned intoXhoI/NotI or XhoI/EcoRI sites of MSCV vector. The coding sequence ofhuman CTLA-4 without a cytoplasmic tail was cloned into an MSCV vectoras well. All vectors were used to generate retrovirus and thentransfected into cell lines 3T3 or CT26 cell lines. Positive cell linetransfectants were sorted out by FACS using specific mAbs or YFP as amarker.

Generation of monoclonal antibodies to human HHLA2. Hybridomas producingmAbs to human HHLA2 were generated by standard techniques fromsplenocytes of HHLA2-Ig-immunized BALB/c mice fused to NSO myelomacells. Four independent clones, 566.1 (IgG1), 351.7 (IgG1), 457.23(IgG1), and 205.1 (IgG1) were selected by ELISA as their mAbs recognizedHHLA2-Ig, but not controls including B7x-Ig, B7-H3-Ig, and normal humanIgG. After this preliminary screening, specificity of mAbs were furtherdetermined by FACS positive staining of a 3T3 transfectant expressingHHLA2-YFP but negative staining of transfectants expressing other humanB7s (B7-1, B7-2, B7g, PD-L1, PD-L2, B7-H3, and B7x) and human CD28family members (CD28, CTLA-4, ICOS, and PD-1). mAbs were purified byProtein G Plus Agarose columns and biotinylated with EZ-LinkSulfo-NHS-Biotin kit (Thermo Scientific).

Antibodies and flow cytometry. Cells were incubated with Fc blockingreagents and then stained with combinations of the following anti-humanantibodies: CD152-PE, CD28-PE, B7-1-PE, B7-2-PE, PD-1-PE, PD-L1-PE,PD-L2-PE, ICOS-PE, ICOSL-PE, B7H4-PE, CD14-FITC, CD19-FITC,CD8a-Percp-Cy5.5, CD4-APC, CD83-APC, streptavidin and isotype controls(eBioscience). Biotinylated anti-hB7-H3 was purchased from R&D. Forreceptor binding, cells were incubated with HHLA2-Ig, B7x-Ig, or controlIg for 45 min on ice and then stained with PE-anti-human IgG Fc (JacksonImmunoresearch). Samples were acquired on a FACSCalibur, LSRII or LSRIIyellow (BD Biosciences), and analyzed with FlowJo (Treestar).

Human antigen-presenting cells and activation. Human CD19+B cells andCD14+ monocytes in PBMCs were activated as previously described (38). Bcells were activated by IFN-γ (100 ng/ml, eBioscience) and LPS (60μg/ml, sigma) for three day, and monocytes were stimulated by IFN-γ (100ng/ml) and LPS (100 ng/ml) for three days. DCs were generated from humanblood monocytes (47). Monocytes from PBMCs were incubated with completedRPMI1640 containing 10% human serum AB (Atlanta Biological), humanGM-CSF (100 ng/ml, R&D) and human IL-4 (50 ng/ml) for six days togenerate immature DCs. These immature DCs were further stimulated withLPS (1 m/ml) and IFN-γ (100 ng/ml) for two days to generate mature DCs.

Human T cell coinhibition assay. Human T cells were purified from PBMCswith CD2 Microbeads (Miltenyi Biotec) and incubated (2×10⁵/well) withdifferent concentrations (0.1-10 μg/ml) of plate-bound anti-CD3 (OKT3,eBioscience) for three days. The T cell proliferation was determined byMTT assay and plates were read at 570 nm. T cells from different donorsneeded different concentrations of anti-CD3 to achieve the middle levelof proliferation. After determining the suitable anti-CD3 concentrationfor each donor T cells, 96-well flat-bottom plates were pre-coated withanti-CD3, HHLA-2-Ig, control Ig, or B7x-Ig in PBS at 4° C. overnight.Wells were washed and incubated with purified T cells for three days. Tcell proliferation was then measured with MTT assay. For the CFSE(Sigma) labeled proliferation assay, CFSE-labeled human T cells wereincubated with plate-bound anti-CD3, HHLA-2-Ig, control Ig, or B7x-Igfor five days, and stained with anti-CD4 and anti-CD8 for flowcytometry.

Cytokine analysis. Aliquots of supernatants were collected at 70 h afterinitiation of T cell cultures. Th1/Th2/Th9/Th17/Th22 13plex FlowCytomixMultiplex (eBioscience) was used for the measurement of human IFN-γ,IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p′70, IL-13, IL-17A,IL-22, and TNF-α according to the manufacturer's instructions.

Confocal microscopy. Cells were seeded on glass bottom microwell dishes(MatTek Corp.) for 48 h and then were observed by using Leica SP2confocal microscopy.

Statistics. Statistical significance was calculated with the paired- orunpaired t-test using Prism software version 4.0b (GraphPad). A p valueof <0.05 was considered statistically significant.

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What is claimed is:
 1. A method of attenuating HHLA2 inhibition of a CD4cell proliferation or of a CD8 T cell proliferation or of a T cellcytokine production in a subject comprising administering an anti-HHLA2antibody, or HHLA2-binding fragment thereof, to the subject.
 2. Themethod of claim 1, wherein the CD4 cell proliferation or a CD8 T cellproliferation is TCR-mediated.
 3. The method of claim 1, wherein the CD4cell proliferation is attenuated or inhibited.
 4. The method of claim 1,wherein the CD8 cell proliferation is attenuated or inhibited.
 5. Themethod of claim 1, wherein the T cell cytokine production is attenuatedor inhibited.
 6. The method of claim 1, wherein the HHLA2 to which theanti-HHLA2 antibody is directed is a human HHLA2 protein.
 7. The methodof claim 6, wherein the HHLA2 protein comprises consecutive amino acidresidues having the sequence set forth in SEQ ID NO:1.
 8. The method ofclaim 1, wherein the antibody is a humanized antibody, a chimericantibody or an isolated human antibody.
 9. The method of claim 1,wherein the antibody is a monoclonal antibody.
 10. The method of claim1, wherein the anti-HHLA2 antibody or HHLA2-binding fragment isconjugated to a cytotoxic agent, an anti-cancer drug or achemotherapeutic agent.